The aim of this research program is to understand in molecular detail the mechanism by which the replication of Herpes Simplex-1 (HSV-1) virus DNA is initiated and sustained. We propose to reconstitute in vitro with purified enzymes whose structure and mechanism we will examine in detail, the orderly, semiconservative replication of the HSV-1 genome. We expect that these studies should provide us with an insight into the mechanism by which replication of a eukaryotic genome is initiated. They should also yield information about the replication of a class of animal viruses that are of great public health importance. The investigation will be organized along the following lines: A. Analysis of purified HSV-1 encoded replication enzymes 1. Origin binding protein (UL9 protein) a. Origin specific helicase activity b. Interaction of UL9 protein with other HSV-1 replication enzymes c. Phosphorylation of LTL9 protein 2. DNA polymerase - UL42 protein complex a. Three dimensional structure of DNA polymerase b. Mechanism of DNA polymerase reaction c. 5' -> 3' exonuclease (RNase H) activity of DNA polymerase d. Phosphorylation of UL42 protein 3. DNA helicase - primase 4. Single-strand DNA binding protein - ICP8 B. Reconstitution of Origin (Ori-s) dependent DNA replication 1. Role of origin binding protein in initiation 2. Specific approaches to reconstitution of Ori-s dependent DNA replication a. Transcriptional activation of initiation b. Requirement for host factors c. Mechanistic studies 3. Regulation of HSV-L DNA replication